Summary/Abstract: G protein Coupled Receptors (GPCRs) transduce information from chemical or hormonal stimuli across the cell membrane to activate heterotrimeric G proteins and thereby elicit physiological/cellular responses. Adhesion G Protein Coupled Receptors (aGPCRs) comprise a 33-member sub-family of GPCRs that have become subjects of intense investigation due to an expanding understanding of the roles these receptors have in developmental disorders and adult cancers. Despite the intense study, direct measurements of heterotrimeric G protein activation by any aGPCR family member had never been made until our very recent work. We leveraged a new technology that we developed to purify large quantities of all classes of heterotrimeric G protein ? subunits and successfully reconstituted G protein activation mediated by two adhesion GPCRs (GPR56 and GPR110). We determined the G protein subtypes that GPR56 and GPR110 activate and found that the extracellular fragments of both receptors inhibit the G protein activating activity of the receptor portion that is intercalated in the membrane (TM or Trans-membrane domain). Receptor activation occurs when the inhibitory extracellular fragment is dissociated from the TM domain, perhaps by the action of protein ligands in the extracellular space that bind to it. This act unmasks a short portion of the TM domain that acts upon itself in positive regulatory fashion (a tethered agonist). These preliminary data lead us to propose that the entire adhesion GPCR class shares a common mechanism of action in being regulated by tethered agonists. We seek to verify the mechanism for GPR56, GPR114, and the Group VI adhesion GPCRs and perform detailed studies to define the exact composition of the tethered agonist and the way that it engages the TM domain. We will conduct studies to investigate the way that natural ligands of the receptors act to reveal the tethered agonist while we molecularly identify authentic receptor ligands. Screens of small molecule chemical libraries will be conducted to identify synthetic adhesion GPCR modulators. These compounds will greatly aid our mechanistic studies as research tools and may find use as leads to block the pathophysiologic actions of adhesion GPCRs in disease contexts. Understanding the mechanism of action of adhesion GPCRs is a pre- requisite to the development of therapies for the increasing number of diseases ascribed to this GPCR-class. Our work strives to elucidate the mechanism of adhesion GPCR action.